Astrocytes are now appreciated as active participants in synaptic function, plasticity and development. However, the study of the astrocytic role in basic synaptic functions has lagged behind that of the synapses themselves. To appreciate fully the role astrocytes play in synaptic function, it is necessary to bring their study to the same level of resolution that has become commonplace in synaptic studies. This implies that we must develop means to study the structure, function, and plasticity of peripheral astrocytic processes (PAPs). PAPs are the points of physical contact between astrocytes and synapses, and as such are likely to be the sites of the critical interactions between them. However, their extremely small size impedes their study in living tissue. The overarching goal of this proposal is to develop methods of studying PAPs optically in intact, living tissue. The experiments described here are designed not only to test these novel techniques, but also to provide essential information about the development and plasticity of PAPs. Not all synapses have direct physical access to an astrocytic process. Astrocytes are capable of detecting extracellular glutamate, and are motile in vitro. Does glutamate released from active synapses influence astrocytic morphology and thereby influence the development, maintenance or plasticity of PAPs? We will address this question by testing the hypotheses that (1) astrocytes are motile in intact tissue, and that motility decreases with postnatal development; (2) astrocytes detect, and respond morphologically to, synaptic activity, and (3) changes in glial morphology are related temporally and spatially to intracellular [Ca2+] transients evoked by local synaptic activity. Astrocytic and synaptic structure and function must be tightly coupled for the brain to function properly. However, it is only by studying astrocytes at the level at which they interact with synapses that we will be able to begin truly to understand this critical relationship.